Production of dextran-dextrinase



Refining Company, New York, N. Y., a-corporation wilNewJersey No Drawing. Application Juni -2451955 serial'Noi 517,913

tion, .gluconic acid, makes the system acidic and thereby.

inhibits enzyme'formation. Accordingly, the maifob'ject of this invention is tohprovide:anaimproved process for making dextran-dextninase. wOther objects will .appear hereinafter.

-5 ductionif dextranldextrinase.

-I have discovered-that polyhydr-icalcohols, :e... g .,'-sori bitbLnramiitol, glycerol, when used asaai's'iibstrate for the ..-production of dextran-dextrinasqg'ive substantially higher T'yields 6f the enzyme. 1N0 substantial amountsbfacid are 'produced'ther'e'fro'm arid separation--of the"enzyme iroin the ketose by-products is simple, e. g., by precipitation with water miscible organic compounds, acetone, alcohol, etc., in the presence of dextran as claimed in co-pending application Serial No. 517,912. The ketose is recovered from the supernatant liquor. The dextran-dextrinase, which is contained in the precipitate, may be used, if desired, for the conversion of partially hydrolyzed amylaceous material to dextran. r

In carrying out the invention, the polyhydric alcohol is mixed with an organic nitrogenous material, "such as yeast extract, corn steep liquor, and the like. This medium is sterilized in conventional manner, the pH adjusted to about 3.5 to about 7.5, and then inoculated with a culture of Acetobacter capsulatum or Acetobacter viscosum. The medium is then incubated under conditions conducive to the formation of dextran-dextrinase. To the liquor may then be added a partially hydrolyzed amylaceous material which is thereupon converted to dextran by the dextrandextrinase. After the conversion to dextran is completed, the dextran is separated from the ketose and residual carbohydrate by precipitation with a water-miscible solvent. The ketose may then be separated from the carbohydrate by evaporation and crystallization. Alternately and preferably, after formation of the dextran-dextrinase and ketose, the dextran-dextrinase may be precipitated and separated. I

The following examples which are typical and not limiting in any sense will further illustrate the invention.

EXAMPLE I Media composed of 0.5 gram of yeast extract per 100 milliliters and of various carbon sources were sterilized,

adjusted to pH 6.0, and inoculated with 10 percent by volume of a culture of Acetobacte'r capsulatum grown 24 hours on 2 percent sorbitol and 0.5 percent yeast extract medium. After fermentation for 16 hours in shaken flasks with aeration, the liquors were adjusted to pH 4.5, 1 percent by volume of toluene was added to prevent oxidation of glucose by the glucose dehydrogenase enzyme present. To 100 milliliters of culture liquor was added 15 grams dry substance of a 17 D. E. acid hydrolyzate of corn starch. The mixtures were then incubated for 16 hours at 28-30 2,833,695 siiintented May 6, 1958 ,-iciivith'constantagitation. The rateof dextranf formation I dextran-dextrinase activity)- was measured. -It is evident able 1) that the polyhydric alcohols, e. ,g., .mannitol, glycerol, and sorbitol, are excellent substrates for proice Table I PRODUCTIQN DEXTRAN-DEXTRINASE ON "DIFFERENT 'CARBOhI SOURCES Oompara-; r ,tive

Dextran- .Dextrinase Activity, percent Amount of Carbonf So'urce, Grams per 100ml.

' "Fermentation :Byslrodubt --Ghrbhn Source Fructose.

gDlhydi-oxyacetone. Sorbose. nGldc'onirra'cid.

Do. Inosose,

!ihearse onhtghen dextroseeon'centiation \irill result in lesser dextrandextrmase formation because ot the excessiveabiditmproduced.

v EXAMPLE II 'Me'dia containing-2g gramsof sorbitol and .025 rgram' if'Of kyeast extr actgper 100 ml. were sterilized, adjusted ito var-ious {pH 'values, andinoculated with a s-ciflturettof "aicetobactencgzpsulatum. :Ait-erfifennentation for: 'l6fhours 0 *with constant ragitat-ion rand aeration, :the illiquors were adjusted no; -4.-5 and .1 ;;percent ==by #volume of-toluene wasiadded. 1

The activity of the culturevliquors='=was=determinedaon starch hydrolyzate. To 100 ml. of each culture liquor was added 15 grams dry substance of a 17 D. E. hydrolyzate of corn starch. The mixtures were then incubated at 2830 6. for 16 hours with constant agitation and the nate of dextran formation determined. A portion of the culture liquor was analyzed for sorbose content.

0 The data (Table I I) show that simultaneous production of sorbose and dextran-dextrinase occurs over a wide range of initial pH values.

EXAMPLE III Media containing 0.5 gram of yeast extract per ml. and various amounts of sorbitol were sterilized, adjusted to pH 6.0, and inoculated with a culture of Acetobacter capsulatum. After fermentation under conditions of constant agitation and aeration, the liquors were analyzed for sorbose content and for dextra'n-dextrinase activity as described above. The results (Table 111) show that sorbose and dextran-dextrin'ase enzyme are simultaneously and efiiciently produced over a wide range of initial sorbitol concentrations.

' Table III Compare Time of tivo Initial Sorbitol Concentration, Fermenta- Conversion Dextran- 1 Grams per 100 m1. tion, to Sorbose, Dextrinase Hours Percent Activity.

' Percent EXAMPLE -IV This example illustrates the separation of dextrandextrinase from the sorbose.

Dextran-dextrinase was produced from sorbitol as described in Example 1. To the culture liquor at pH 4.5 was added one percent dextran and 25 to 30 percent of acetone. to precipitate the enzyme. The precipitate was dissolved in water and used in the production of dextran from starch hydrolyzate as described in Example 1. The supernatant liquor from the precipitation of the enzyme contained 98 percent sorbose based on the Weight of sorbitol added. From the enzyme-converted starch hydrolyzate was obtained 35 percent dextran. based on the weight of starch hydrolyzate added.

I claim:

1. In the production of dextran-dextrinase and ketoses by fermentation of a nutrient solution by a dextrandextrinase producing organism, the improvement which comprises increasingthe' yield of dextr-an-dextrinase by dextran-dextrinase therefrom.

2. The method of producing dextran-dextrinase and ketoses which comprises increasing the yield of dextrandextrinase by culturing an organism from the group consisting of Acetobacter capsulatum and Acetobacler viscasum in a nutrient medium containing an added polyhydric alcohol as the source of carbon at a pH within the range of about 3.5 to 7.5 until a substantial amount of enzyme is produced and recovering dextran-dextrinase therefrom.

3. Process according to claim 1 wherein the polyhydric alcohol is sorbitol.

4. Process according to claim 1 wherein the polyhydric alcohol is mannitol.

5. Process according to claim 1 wherein the polyhydric alcohol is glycerol.

6. Process according to claim 2 wherein the polyhydric alcohol is sorbitol.

7. Process according to claim 2 wherein the polyhydric alcohol is mannitol.

8. Process according to claim 2 wherein the polyhydric alcohol is glycerol.

References Cited in the file of this patent UNITED STATES PATENTS Kooi Sept. 21, 1954 OTHER REFERENCES I A..J. Salle, publ. by McGraw-Hill Book Co. Inc. of New York, pp. 3-81 to 383. 

2. THE METHOD OF PRODUCING DEXTRAN-DEXTRINASE AND KETOSES WHICH COMPRISES INCREASING THE YIELD OF DEXTRANDEXTRINASE BY CULTURING AN ORGANISM FROM THE GROUP COMSISTING OF ACETOBACTER CAPSULATUM AND ACETOBACTER VISCOSUM IN A NUTRIENT MEDIUM CONTAINING AN ADDED POLYHYDRIC ALCOHOL AS THE SOURCE OF CARBON AT A PH WITHIN THE RANGE OF ABOUT 3.5 TO 7.5 UNTIL A SUBSTANTIAL AMOUNT OF ENZYME IS PRODUCED AND RECOVERING DEXTRAN-DEXTRINASE THEREFROM. 